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钴胺素依赖的蛋氨酸合酶催化N5-甲基四氢叶酸转移甲基至同型半胱氨酸生成蛋氨酸和四氢叶酸,直接参与蛋氨酸循环、叶酸循环及含硫氨基酸代谢,与DNA、蛋白质合成及生物甲基化有密切关系。本研究采用蛋白层析技术,将大鼠肝匀浆经超声破碎和高速离心处理后,依次经过DE-52批处理、Q Sepharose Fast Flow离子交换层析和CHT陶瓷羟基磷灰石吸附柱层析进行纯化,并对纯化产物进行了SDS-PAGE和Western blotting鉴定。采用分光光度法测定蛋氨酸合酶的活性,对纯化酶的酶促反应动力学进行了研究,确定了最佳反应条件,动力学结果显示蛋氨酸合酶的双底物酶促反应的机制为乒乓机制。研究表明,采用层析技术纯化得到的蛋氨酸合酶适用于以其为靶点的化合物高通量筛选。
Cobalamin-dependent methionine synthase catalyzes the N5-methyltetrahydrofolate transfer of methyl to homocysteine to methionine and tetrahydrofolate, which is directly involved in methionine cycle, folic acid cycle and sulfur-containing amino acid metabolism, and DNA, protein synthesis And biological methylation are closely related. In this study, the protein of rat liver homogenate by ultrasonication and high-speed centrifugation, followed by DE-52 batch processing, Q Sepharose Fast Flow ion exchange chromatography and CHT ceramic hydroxyapatite column chromatography The purified product was identified by SDS-PAGE and Western blotting. The activity of methionine synthase was determined by spectrophotometry, the enzymatic kinetics of purified enzyme was studied, and the optimum reaction conditions were determined. The kinetic results showed that the mechanism of enzymatic reaction of double-substrate of methionine synthase was ping-pong mechanism . Studies have shown that the methionine synthase purified by chromatography is suitable for high-throughput screening of the target compounds.